Nucleic acid aptamer screening method for inducing in-situ phase separation by using lanthanide series metal and application of nucleic acid aptamer screening method

Abstract

The invention discloses a nucleic acid aptamer screening method utilizing lanthanide series metal to induce in-situ phase separation and application, relates to the technical field of nucleic acid aptamer screening, and successfully develops a PS-SELEX method by utilizing lanthanide series metal ions Gd < 3 + > to effectively separate nucleic acid aptamers in a solution from a random single-stranded DNA library through phase separation. The method effectively improves library fixation and efficiency in a nucleic acid aptamer screening process, further takes Cd < 2 + > and Fe < 2 + > as research objects, adopts a PS-SELEX technology, utilizes a random single-stranded DNA library and Gd < 3 + > to form nanoparticles through self-assembly, realizes in-situ phase separation of the nucleic acid aptamer library, and realizes in-situ phase separation of the nucleic acid aptamer library through multiple rounds of forward screening and reverse screening of corresponding interfering ions. Two nucleic acid aptamer sequences capable of specifically recognizing Cd < 2 + > and Fe < 2 + > respectively are obtained through screening, and a theoretical basis is provided for efficient screening of high-affinity nucleic acid aptamers.

Description

Technical Field

[0001] The present invention relates to the technical field of aptamer screening, and particularly relates to a method for screening aptamers by inducing in-situ phase separation with lanthanide metals and its application. Background Art

[0002] An aptamer is a segment of oligonucleotide with a targeting function, mainly obtained by in vitro screening through the Systematic evolution of ligands by exponential enponential enrichment (SELEX) technology. Due to its high affinity, easy modification, and low immunogenicity, it has become an important direction for antibody substitutes. However, how to screen aptamers with high affinity and high selectivity is currently the main problem hindering the application of aptamers.

[0003] Traditional screening methods, such as the magnetic bead method, rely on target immobilization and require complex modification of small molecules (especially metal ions) to be fixed on the magnetic beads. Due to the difficulty of target immobilization, it may lead to distortion of the target structure and an increase in screening deviation. Moreover, the random library may non-specifically bind to the stationary phase carrier through hydrophobic interaction / electrostatic adsorption, and repeated washing is required to reduce background noise, resulting in the loss of aptamer sequences. To avoid the problem of target immobilization, improved methods with library immobilization as the core have been developed in recent years, such as the GO-SELEX technology that screens based on the π-π adsorption difference of ssDNA by graphene oxide (GO). However, the adsorption ability of graphene oxide for double-stranded DNA is significantly weaker. If the ssDNA rich in stem-loop structures forms local double strands, the adsorption efficiency will decrease, and the surface oxidation degree and number of layers of GO are not uniform, resulting in fluctuations in adsorption performance between batches. Therefore, there is an urgent need for a simple, highly stable, high-throughput method for screening aptamers that can efficiently and quickly immobilize the library.

[0004] The coordination chemistry of metal ions with DNA provides a novel and promising strategy for the design and synthesis of functional DNA nanomaterials. Through direct coordination-driven self-assembly, metal-DNA nanostructures with adjustable and controllable properties can be precisely synthesized. Due to their high coordination number and flexibility, lanthanide metals are regarded as excellent ligands for self-assembly with nucleic acids and have been widely used in drug delivery, bioimaging, and enhancing enzyme reactions. However, there is currently no precedent for using this property of lanthanide metals and DNA for aptamer screening. Summary of the Invention

[0005] In view of the technical problems existing in the above-mentioned existing research and technologies, the purpose of the present invention is to provide a method for screening nucleic acid aptamers by inducing in-situ phase separation with lanthanide metals (denoted as PS-SELEX), specifically relating to a method for realizing rapid library immobilization based on the coordination self-assembly of lanthanide metal ions (such as Gd 3+ ), and combining with the ion competition effect to efficiently screen high-affinity and high-specificity nucleic acid aptamers.

[0006] The present invention realizes the above purpose through the following technical solutions: The first object of the present invention is to provide a method for screening nucleic acid aptamers by inducing in-situ phase separation with lanthanide metals, including the following steps: (1) Synthesize a random single-stranded DNA library and primer sequences; (2) Incubate the random single-stranded DNA library with lanthanide metal ions to form lanthanide metal-nucleic acid aptamer self-assembled nanoparticles, centrifuge to collect the precipitate, and wash the formed self-assembled nanoparticles to remove excess lanthanide metal ions; (3) Positive screening: Incubate the lanthanide metal-nucleic acid aptamer self-assembled nanoparticles with target metal ions, and recover the library that can bind to the target metal ions; (4) Using the recovered library as a template, perform PCR amplification with primer sequences to obtain a PCR amplification product; (5) Prepare a secondary library from the PCR amplification product, use the secondary library as a screening library, and perform multiple rounds of screening according to the process of steps (2) to (5); (6) Introduce interfering metal ions for reverse screening in a single round of the multiple rounds of screening; (7) After multiple rounds of screening, perform high-throughput sequencing analysis on the final-round secondary library, select sequences for affinity verification, and according to the affinity verification results, screen out nucleic acid aptamer sequences with high affinity for the target metal ions.

[0007] As a further optimization scheme of the present invention, the lanthanide metal ion is Gd 3+ , and the nucleic acid library is self-assembled with Gd 3+ to form nanoparticles (denoted as Gd-DNA nanoparticles), realizing in-situ phase separation of the nucleic acid aptamer library. The formed Gd-DNA nanoparticles have a diameter of dozens of nanometers.

[0008] As a further optimization scheme of the present invention, the interfering metal ions are a mixture of other metal ions except the target metal ions. Further, when screening Cd 2+ nucleic acid aptamers and Fe 2+ nucleic acid aptamers, the interfering metal ions include Mn 2 + , Cu2+ , Ca 2+ , Al 3+ , Ba 2+ , Cd 2+ , Ni 2+ , Zn 2+ , Ce 3+ , Pb 2+ , Cr 3+ , Fe 3+ and Hg 2+ mixture.

[0009] As a further optimization scheme of the present invention, the target metal ion is Cd 2+ , screening the random single-stranded DNA library for Cd 2+ aptamer has the sequence of 5'-ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACTA-3', where N 40 represents a sequence composed of 40 arbitrary nucleobases. During screening, the 5'-end of the random single-stranded DNA library sequence was further modified with a FAM fluorescent group. The primer sequences for screening the Cd 2+ aptamer include the forward primer: 5'-ATGTCTACTAGCGTAGCATC-3' and the reverse primer: 5'-TAGTCCATGTAACGGTACGA-3'. During screening, the 5'-end of the forward primer was further modified with a FAM fluorescent group, and the 5'-end of the reverse primer was further modified with biotin Biotin.

[0010] As a further optimization scheme of the present invention, the target metal ion is Fe 2+ , screening the random single-stranded DNA library for Fe 2+ aptamer has the sequence of 5'-GACGTGATCTGACTCAAGCT-N 40 -GATTCGACAGGCTCTAGAGT-3', where N 40 represents a sequence composed of 40 arbitrary nucleobases. During screening, the 5'-end of the random single-stranded DNA library sequence was further modified with a FAM fluorescent group. The primer sequences for screening the Fe 2+ aptamer include the forward primer: 5'-GACGTGATCTGACTCAAGCT-3' and the reverse primer: 5'-ACTCTAGAGCCTGTCGAATC-3'. During screening, the 5'-end of the forward primer was further modified with a FAM fluorescent group, and the 5'-end of the reverse primer was further modified with biotin Biotin.

[0011] As a further optimization scheme of the present invention, screening for Cd 2+When screening for aptamers, the total number of rounds of multiple rounds of screening is 9 rounds, and the rounds of introducing interfering metal ions for counter-selection are the 4th, 6th, and 8th rounds.

[0012] As a further optimization scheme of the present invention, when screening for Fe 2+ aptamers, the total number of rounds of multiple rounds of screening is 10 rounds, and the rounds of introducing interfering metal ions for counter-selection are the 4th, 6th, and 9th rounds.

[0013] The second object of the present invention is to also provide an application of the screening method as described in any one of the above in screening for metal ion aptamers.

[0014] As a further optimization scheme of the present invention, the metal ion aptamer is a Cd 2+ aptamer, and the nucleotide sequence of the Cd 2+ aptamer is as shown in SEQ ID NO.11, and the dissociation constant K 2+ of the Cd 2+ aptamer binding to Cd d is 141 μM.

[0015] As a further optimization scheme of the present invention, the metal ion aptamer is a Fe 2+ aptamer, and the nucleotide sequence of the Fe 2+ aptamer is as shown in SEQ ID NO.12, and the dissociation constant K 2+ of the Fe 2+ aptamer binding to Fe d is 91 μM.

[0016] The third object of the present invention is to also provide an application of the metal ion aptamer as described in any one of the above in preparing a product for specifically recognizing a corresponding target metal ion.

[0017] The present invention has the following beneficial effects: The present invention effectively separates aptamers in solution from a random single-stranded DNA library through phase separation (assembling into Gd-DNA nanoparticles) using the lanthanide metal ion Gd 3+ , successfully develops the PS-SELEX method, effectively improves the library immobilization and efficiency in the aptamer screening process, and provides a new scheme for the efficient screening of aptamers.

[0018] Furthermore, the present invention takes metal ions Cd 2+ and Fe 2+ as research objects, adopts the PS-SELEX technology carried out in the presence of Gd 3+ , and utilizes the nucleic acid library and Gd 3+Self-assembled to form nanoparticles, realizing in-situ phase separation of the aptamer library. Through multiple rounds of forward screening and reverse screening of the corresponding interfering ions, two high-affinity aptamer sequences that can specifically recognize Cd 2+ and Fe 2+ were screened. The metal-ion aptamers screened by the above screening method provide an application basis for the preparation of products that specifically recognize the corresponding target metal ions. Description of the Drawings

[0019] Figure 1 SEM characterization diagram of the Gd-DNA nanoparticles provided in Example 1; Figure 2 TEM characterization diagram of the Gd-DNA nanoparticles provided in Example 1; Figure 3 HAADF-STEM characterization diagram of the Gd-DNA nanoparticles provided in Example 1; Figure 4 Adsorption isotherm of the interaction between GO and ssDNA with different structures provided in Example 2; Figure 5 Kinetic curve of the interaction between GO and ssDNA with different structures provided in Example 2; Figure 6 Adsorption isotherm of the self-assembly of Gd 3+ and ssDNA with different structures provided in Example 2; Figure 7 Kinetic curve of the self-assembly of Gd 3+ and ssDNA with different structures provided in Example 2; Figure 8 Schematic diagram of the screening process provided in Example 3; Figure 9 Affinity determination of Cd-1 and Cd 2+ provided in Example 3; Figure 10 Secondary structure prediction of Cd-1 provided in Example 3; Figure 11 Affinity determination of Fe-1 and Fe 2+ provided in Example 3; Figure 12 Secondary structure prediction of Fe-1 provided in Example 3; Figure 13 cDNA length and ratio optimization provided in Example 4. In the figure, a-b corresponds to Cd 2+ ion detection; c-d corresponds to Fe 2+ ion detection; Figure 14The fluorescence detection curves of Cd-1 provided in Example 4 with different Cd 2+ concentrations; Figure 15 The fluorescence detection curves of Fe-1 provided in Example 4 with different Fe 2+ concentrations; Figure 16 Specificity verification of the selected Cd 2+ aptamers provided in Example 4; Figure 17 Specificity verification of the selected Fe 2+ aptamers provided in Example 4. Detailed implementation manners

[0020] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following specific implementation manners are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0021] In the following examples, the specific experimental methods not specified can be carried out according to the conventional methods. The materials, reagents, etc. used, unless otherwise specified, can be obtained through commercial channels.

[0022] Example 1. Formation of nanoparticles by the self-assembly of Gd 3+ ions and DNA The method for forming nanoparticles by the self-assembly of Gd 3+ ions and DNA provided in this example is described, and the morphology of the nanoparticles is characterized. The specific steps are as follows: 1. Preparation of Gd-DNA nanoparticles Synthesize a DNA library with a sequence length of 80 nt. The sequence of the DNA library is ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACT (SEQ ID NO.5). Prepare Gd-DNA nanoparticles by mixing 300 nmol of Gd 3+ with 100 pmol of ssDNA library in 100 μL of binding buffer (the molar ratio of Gd 3+ to ssDNA is 3000:1). The binding buffer contains 50 mM HEPES, 100 mM NaCl, 2 mM MgCl2, and 0.025% Tween-20, and is adjusted to pH 7.5. After incubating at room temperature for 3 h, wash the Gd-DNA nanoparticles several times to remove the excess Gd 3+ , and then use them for further characterization.

[0023] 2. Characterization of Gd-DNA nanoparticles As Figure 1 and Figure 2 shown, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the morphology of Gd-DNA nanoparticles with a size of about dozens of nanometers. Energy-dispersive X-ray spectroscopy (EDX) elemental mapping was used to confirm the uniform distribution of phosphorus (P) and nitrogen (N) from the DNA library and gadolinium (Gd) elements within the nanoparticles, as Figure 3 shown.

[0024] Example 2. Comparison of Gd 3+ -induced DNA self-assembly and GO-mediated DNA immobilization efficiency The method for systematically comparing the performance of PS-SELEX and GO-SELEX provided in this example compares the immobilization efficiency of the two methods on DNA libraries with different secondary structures, and specifically includes the following steps: 1. Synthesis of DNA libraries with different secondary structures Four DNA libraries with a length of 80-nt each but different secondary structures were synthesized, as shown in Table 1.

[0025] Table 1. Sequence information of DNA libraries with different secondary structures ; From Structure 1 to Structure 4, bases gradually form complementary pairs along the nucleotide chain, and the hybridization process significantly increases the rigidity of the DNA structure.

[0026] 2. Testing the adsorption capacity of GO and Gd 3+ for ssDNA with different structures The adsorption capacity of GO and Gd 3+ for 100 pmol of DNA strands with different secondary structures was further evaluated. These two reagents were used to bind to the DNA library at different concentrations and exposure times. After incubation at room temperature, the supernatant was centrifuged to separate the unbound DNA strands. Subsequently, 1×SYBR Gold dye was introduced, and the fluorescence intensity (F) and the initial value (F0) were measured. The adsorption capacity was calculated using the following formula: Adsorption capacity = (F0 - F) / F0 × 100%. The test results are as Figures 4 - 7 shown.

[0027] As the bases gradually form complementary pairs along the nucleotide chain, the hybridization process significantly increases the rigidity of the DNA structure, and the enhanced rigidity reduces the adsorption capacity of GO on the DNA strand from 99.8% to 69.6% ( Figure 5), which is due to the reduced availability of flexible sites for π-π stacking interactions, resulting in the preferential adsorption and separation of enriched sequences during GO-SELEX. In contrast, due to different immobilization mechanisms, the self-assembly efficiency of Gd 3+ with DNA strands is not affected by the secondary structure of the nucleic acid strands ( Figure 6 ).

[0028] The adsorption isotherm curves of various DNA strands immobilized by Gd 3+ show that all strands achieve nearly complete adsorption at higher concentrations, although the adsorption efficiency varies at lower Gd Figure 7 concentrations. This behavior is significantly different from the adsorption curve of GO for nucleic acids ( 3+ ). The kinetic curves of Gd Figure 4 -mediated DNA strand immobilization demonstrate the rapid and efficient binding of different DNA strands within just a few minutes. These results confirm that the pool immobilized by Gd 3+ is significantly more efficient and exhibits less batch variation compared to traditional substrates such as magnetic beads or graphene. 3+

[0029] Example 3. Screening for Cd 3+ ions and Fe 2+ ions nucleic acid aptamers using Gd 2+ to induce in-situ phase separation The method for screening Cd 3+ ions and Fe 2+ ions nucleic acid aptamers using Gd 2+ to induce in-situ phase separation provided in this example is shown in the schematic diagram of the screening process as Figure 8 shown, and specifically includes the following steps: 1. Synthesize a random single-stranded DNA library and primer sequences The full length of the random single-stranded DNA library is 80 bp, including 20 bp of fixed sequence bases at both ends and 40 bp of random sequence bases in the middle. When performing the following screening method, a FAM fluorescent group was further modified at the 5' end of the random single-stranded DNA library sequence.

[0030] (1) For screening Cd 2+ nucleic acid aptamers: The initial library is: 5'-ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACTA-3' (denoted as library-1, SEQ ID NO.5), where N 40 represents a sequence composed of 40 arbitrary nucleotide bases linked together.

[0031] ​The primer sequences include a forward primer: 5’-ATGTCTACTAGCGTAGCATC-3’ (SEQ ID NO.6); and a reverse primer: 5’-TAGTCCATGTAACGGTACGA-3’ (SEQ ID NO.7). When performing the following screening method, the 5’ end of the forward primer was further modified with a FAM fluorophore, and the 5’ end of the reverse primer was further modified with biotin.

[0032] (2) For the screening of Fe 2+ Nucleic acid aptamer: The initial library was: 5’-GACGTGATCTGACTCAAGCT-N 40 -GATTCGACAGGCTCTAGAGT-3’ (denoted as library-2, SEQ ID NO.8), where N 40 represents a sequence composed of 40 arbitrary nucleotide bases linked together.

[0033] The primer sequences include a forward primer: 5’-GACGTGATCTGACTCAAGCT-3’ (SEQ ID NO.9); and a reverse primer: 5’-ACTCTAGAGCCTGTCGAATC-3’ (SEQ ID NO.10). When performing the following screening method, the 5’ end of the forward primer was further modified with a FAM fluorophore, and the 5’ end of the reverse primer was further modified with biotin.

[0034] 2. First round of screening 2.1. Preparation of Gd-DNA nanoparticles Dissolve 500 pmol of the DNA library and 300 nmol of Gd 3+ in the binding buffer (50 mM HEPES, 100 mM NaCl, 2 mM Mg 2+ , 0.025% Tween-20, pH 7.5), and rotate and incubate at room temperature for 60 min to obtain Gd-DNA self-assembled nanoparticles.

[0035] 2.2. Positive screening Add 500 nmol of Cd 2+ ions or Fe 2 + ions to the Gd-DNA nanoparticle suspension formed in step 2.1, and rotate and incubate at room temperature for 60 min to allow the Cd 2+ ions or Fe 2+ ions to competitively bind out from the Gd-DNA self-assembled nanoparticles the ones that pair with Cd 2+ ions or Fe 2+Nucleic acid aptamers with affinity for ions. Centrifuge at 15,000 rpm for 10 min and retain the supernatant to obtain a library that can bind to Cd 2+ ions or Fe 2+ ions.

[0036] 2.3. PCR Amplification Use the library in the supernatant obtained in step 2.2 as a template for PCR amplification. The total PCR system is 50 μL in volume (including 1 μL of the recovered secondary library, 25 μL of 2×Taq PCR Mix, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, and made up to a final volume of 50 μL with DEPC H2O). The amplification conditions are: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 60 s, for a total of 20 cycles, final extension at 72°C for 3 min, and incubation at 4°C.

[0037] 2.4. Preparation of Secondary Library Pipette 50 μL of streptavidin magnetic beads onto a magnetic rack, let stand for 1 min, remove the supernatant, add 200 μL of 1×PBS buffer (containing 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, 1 mg / mL BSA) to the centrifuge tube, rotate and incubate for 60 min, and perform magnetic separation. Wash three times with 200 μL of 1×PBS buffer, add the PCR product prepared in step 2.3, add one-third volume of 4 M NaCl solution, place the centrifuge tube on a rotary mixer, and incubate with shaking at room temperature for 60 min. Place the centrifuge tube back on the magnetic rack, let stand for 1 min, aspirate and discard the supernatant, and wash the magnetic beads three times with 200 μL of 1×PBS buffer. Add 30 μL of 0.2 M NaOH solution, mix for 1 min to denature the double-stranded DNA. Place the centrifuge tube on the magnetic rack, let stand for 1 min, aspirate the supernatant into a new centrifuge tube, adjust the pH of the library to neutral with hydrochloric acid, and use it as the secondary library for the next round of screening and measure its concentration.

[0038] 3. Counter-Screening Different from incubating Cd 2+ ions or Fe 2+ ions with Gd-DNA nanoparticles in the first round of screening: During counter-screening, add interfering ions (including Mn 2+ , Cu 2+ , Ca 2+ , Al 3+ , Ba 2+ , Ni 2+ , Zn 2 + , Ce3+ , Pb 2+ , Cr 3+ , Fe 3+ and Hg 2+ were mixed (the dosage of all the above ions was 100 nmol), incubated with the library for 60 min, and the supernatant was removed by centrifugation to remove the library bound to the interferents. The remaining Gd-DNA nanoparticles were continued with the positive screening step.

[0039] 4. Multiple rounds of screening (1) For Cd 2+ Ion aptamer screening: Replace the initial library in the first round of screening with the secondary library, repeat the screening process, adjust the screening pressure according to the recovery rate of each round of screening, and introduce counter screening in the 4th, 6th, and 8th rounds of screening. The specific screening conditions are shown in Table 2 below. The library of the 9th round was used for high-throughput sequencing.

[0040] (2) For Fe 2+ Ion aptamer screening: Replace the initial library in the first round of screening with the secondary library, repeat the screening process, adjust the screening pressure according to the recovery rate of each round of screening, and introduce counter screening in the 4th, 6th, and 9th rounds of screening. The specific screening conditions are shown in Table 3 below. The library of the 10th round was used for high-throughput sequencing.

[0041] Table 2. PS-SELEX screening conditions for Cd 2+ ; ; Table 3. PS-SELEX screening conditions for Fe 2+ ; ; 5. Sequencing and sequence analysis After the last round of screening, the enriched ssDNA library was sent for cloning and sequencing. Finally, we selected the sequences with the highest enrichment degree as the representatives for subsequent characterization. The two sequences obtained by screening were respectively: Cd-1: 5’-ATGTCTACTAGCGTAGCATCCAACTGGATGCAGATTACAATCTCGAAACTAACTATCATTTCGTACCGTTACATGGACTA-3’ (SEQ ID NO.11); Fe-1: 5’-GACGTGATCTGACTCAAGCTTACCTCTTCAGTTCCTAGGTATTCTAATCTATTTCCTCCCGATTCGACAGGCTCTAGAGT-3’ (SEQ ID NO.12).

[0042] 6. Verification of the Affinity of Aptamers Screened by PS-SELEX for Target Ions (1) Verification of the affinity of the screened aptamer for Cd 2+ aptamer for Cd 2+ ions: ITC technology was used to quantitatively evaluate the binding affinity between the selected aptamer and the target metal ion. Titration was performed using MicroCal PEAQ-ITC. 20 μM Cd-1 was added to the reaction cell, and 2 mM CdCl2 was added to the syringe. The experiment was carried out at a stirring speed of 750 rpm and maintained at 25 °C. The titration experiment consisted of a total of 19 injections, with each injection delivering 2 μL of the metal solution, except for the first injection, which was 0.4 μL. A 120-second interval was maintained between each injection to allow for complete thermal equilibrium. The data was integrated and analyzed using MicroCal PEAQ-ITC Analysis software.

[0043] The final results are as Figure 9 shown. The dissociation constant Kd = 141 μM was obtained, the stoichiometry of the reaction was approximately 8.25, the enthalpy change ΔH was -4.48 kcal·mol -1 , and the entropy change ΔS was 2.60 cal·mol -1 ·K -1 (free energy change -TΔS = -0.775 kcal·mol -1 , 298 K). The secondary structure of Cd-1 was simulated using M-fold, as Figure 10 shown.

[0044] (2) Verification of the affinity of the screened aptamer for Fe 2+ aptamer for Fe 2+ ions: ITC technology was used to quantitatively evaluate the binding affinity between the selected aptamer and the target metal ion. Titration was performed using MicroCal PEAQ-ITC. 50 μM Fe-1 was added to the reaction cell, and 5 mM FeCl2 was added to the syringe. The experiment was carried out at a stirring speed of 750 rpm and maintained at 25 °C. The titration experiment consisted of a total of 19 injections, with each injection delivering 2 μL of the metal solution, except for the first injection, which was 0.4 μL. A 120-second interval was maintained between each injection to allow for complete thermal equilibrium. The data was integrated and analyzed using MicroCal PEAQ-ITC Analysis software. The final results are as Figure 11 shown. The dissociation constant Kd = 91 μM was obtained, the stoichiometry of the reaction was approximately 7.31, the enthalpy change ΔH was -0.351 kcal·mol -1, the entropy change ΔS is 17.3 cal·mol -1 ·K -1 (the free energy change -TΔS = -5.16 kcal·mol -1 , 298K), the secondary structure of Fe-1 was simulated using M-fold, as Figure 12 shown.

[0045] Example 4. Construction of a standard displacement fluorescence assay for specific detection of Cd 2+ and Fe 2+ using the selected aptamers This example provides a method for specifically detecting Cd 2+ and Fe 2+ in solution by using the selected aptamers Cd-1 and Fe-1, which specifically includes the following steps: 1. Modify the aptamers and construct complementary strands of different lengths The two aptamers were modified with FAM fluorescent groups at the 5' end. In addition, complementary cDNA strands with lengths of 14-nt, 16-nt, 18-nt, 20-nt, and 22-nt were constructed respectively, and Dabcyl was modified at the 3' end of the complementary cDNA strands of different lengths.

[0046] (1) For the Cd-1 aptamer, the sequences of the complementary cDNA strands of different lengths are shown in Table 4: Table 4. Sequence information of the complementary cDNA strands constructed for the Cd-1 aptamer ; (2) For the Fe-1 aptamer, the sequences of the complementary cDNA strands of different lengths are shown in Table 5: Table 5. Sequence information of the complementary cDNA strands constructed for the Fe-1 aptamer ; 2. Optimize the length and dosage of the cDNA for fluorescence detection of Cd 2+ or Fe 2+ Incubate 0.1 μM FAM-aptamer with the target ions (Cd at 100 μM, Fe 2+ at 60 μM) for 30 min. Subsequently, introduce complementary strands of different lengths (14, 16, 18, 20, and 22 nucleotides) modified with Dabcyl at a concentration of 0.5 μM. Measure the fluorescence intensity after 20 min of incubation. In addition to optimizing the length of the complementary strand, the present invention also optimized the ratio of the aptamer to its corresponding complementary strand. 2+ Finally, the scheme determined by the present invention is as

[0047] shown in Figure 13As shown, for the detection of Cd 2+ ions, the optimal cDNA strand length is 20-nt, and the optimal ratio of quencher strand to FAM-aptamer is determined to be 5:1; for the detection of Fe 2+ ions, the optimal cDNA strand length is 18-nt, and the optimal ratio of quencher strand to FAM-aptamer is determined to be 3:1.

[0048] 3. Detection of different concentrations of targets using standard displacement fluorescence analysis Using the optimized conditions, incubate the FAM-labeled aptamer with different concentrations of target ions for 30 min. Then, measure the fluorescence intensity (denoted as F) after 20 min in the presence of the quencher strand. After hybridization of the aptamer with the complementary DNA strand, measure the baseline fluorescence intensity, denoted as F0. To determine the binding affinity and detection limit of the assay, calculate the change in fluorescence intensity using the formula (F - F0) / F0. Plot this value against the concentration of the target ion, allowing the calculation of the Kd of the aptamer. Additionally, based on this data, establish the detection limit of the relevant assay to gain insight into the sensitivity and performance of the aptamer-based detection system for the target ion.

[0049] The final results are as shown in Figure 14 and Figure 15 : Similar to ITC, the Kd value of Cd-1 is 122 μM, and the Kd value of Fe-1 is 116 μM. The detection limit for both target ions is 0.3 μM.

[0050] 4. Specificity evaluation of the detection method Expose the FAM-labeled aptamer to 12 potential interfering ions, each at a concentration of 100 μM. As shown in Figure 16 and Figure 17 : Both the Cd-1 and Fe-1 nucleic acid aptamers show the highest fluorescence signals in the presence of their respective target ions, demonstrating high specificity and affinity for their target metal ions (i.e., Cd 2+ , Fe 2+ ).

[0051] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for screening nucleic acid aptamers by inducing in-situ phase separation using lanthanide metals, characterized in that, Comprising the following steps: (1) Synthesize a random single-stranded DNA library and primer sequences; (2) Incubate the random single-stranded DNA library with lanthanide metal ions to form lanthanide metal-nucleic acid aptamer self-assembled nanoparticles; (3) Positive screening: Incubate the lanthanide metal-nucleic acid aptamer self-assembled nanoparticles with target metal ions and recover the library that can bind to the target metal ions; (4) Using the recovered library as a template, perform PCR amplification with the primer sequences to obtain a PCR amplification product; (5) Prepare a secondary library from the PCR amplification product, use the secondary library as a screening library, and perform multiple rounds of screening according to the process of steps (2) to (5); (6) Introduce interfering metal ions for reverse screening in a single round of the multiple rounds of screening; (7) After the multiple rounds of screening are completed, perform high-throughput sequencing analysis on the secondary library of the final round, select sequences for affinity verification, and according to the affinity verification results, screen out nucleic acid aptamer sequences with high affinity for the target metal ions.

2. The method for screening nucleic acid aptamers by inducing in-situ phase separation using lanthanide metals according to claim 1, wherein: The lanthanide metal ion is Gd 3+ .

3. A method for screening nucleic acid aptamers using lanthanide metal-induced in-situ phase separation according to claim 2, characterized in that: The interfering metal ions are a mixture of other metal ions other than the target metal ions, and the other metal ions include Mn 2+ , Cu 2+ , Ca 2+ , Al 3+ , Ba 2+ , Cd 2+ , Ni 2+ , Zn 2+ , Ce 3+ , Pb 2+ , Cr 3+ , Fe 3+ and Hg 2+ .

4. A method for screening nucleic acid aptamers by inducing in-situ phase separation using lanthanide metals according to claim 3, wherein The target metal ion is Cd 2+ , screening for Cd 2+ The sequence of the random single-stranded DNA library of the aptamer for Cd is 5'-ATGTCTACTAGCGTAGCATC-N 40 -TCGTACCGTTACATGGACTA-3', where N 40 represents a sequence formed by connecting 40 arbitrary nucleotide bases.

5. A method for screening nucleic acid aptamers by inducing in-situ phase separation using lanthanide metals according to claim 3, characterized in that The target metal ion is Fe 2+ , screening for Fe 2+ The sequence of the random single-stranded DNA library of the nucleic acid aptamer is 5'-GACGTGATCTGACTCAAGCT-N 40 -GATTCGACAGGCTCTAGAGT-3', where N 40 represents a sequence formed by connecting 40 arbitrary nucleotide bases.

6. Use of the screening method according to any one of claims 1-5 in screening metal ion nucleic acid aptamers.

7. A metal ion nucleic acid aptamer, characterized in that, Obtained by screening using the screening method according to any one of claims 1-5.

8. The metal ion nucleic acid aptamer according to claim 7, wherein The metal ion aptamer is a Cd 2+ aptamer, and the nucleotide sequence of the Cd 2+ aptamer is as shown in SEQ ID NO.

11.

9. The metal ion nucleic acid aptamer according to claim 7, characterized in that, The metal ion aptamer is an Fe 2+ aptamer, and the nucleotide sequence of the Fe 2+ aptamer is as shown in SEQ ID NO.

12.

10. Use of a metal ion nucleic acid aptamer according to any one of claims 7-9 in the preparation of a product for specifically recognizing a corresponding target metal ion.

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